Biology Reference
In-Depth Information
TABLE 13.3
Relative Retention Times in RP-HPLC of Various Phospholipids
in The Most Commonly Used Mobile Phases Acetonitrile/Water
and Propan-2-ol/water. Retention Times Increase from Top to
Bottom.
Acetonitrile/water
Propan-2-ol/water
PA
CL
CL
PE
PI
PI
PS
PS
PE
PA
PC
PC
SM
SM
Lyso PC
Lyso PC
indicated by the detector is referred to as the retention time and is characteristic of the analyte
under conditions of the experiment (e.g. stationary and mobile phase, column length, pres-
sure, and temperature). Initially the HPLC stationary phase was silica gel and the separation
process resembled that described above for TLC. The more polar analytes bind better to the
silica gel while the more non-polar analytes partition more readily into the non-polar mobile
phase and so reach the detector quicker and have a shorter retention time.
While silica gel HPLC is termed 'normal phase' HPLC, it is not the most commonly used
procedure. Reversed phase HPLC (RP-HPLC or RPC) is by far the most popular type of
HPLC used in biological membrane studies
[25
27]
. RP-HPLC employs a non-polar
stationary phase and a polar mobile phase that contains water. The RP-HPLC stationary
phase is often silica gel that has been modified by RMe
2
SiCl where R is either a C-8 or
C-18 saturated hydrocarbon chain. Therefore the RP-HPLC stationary phase is polar silica
with an oily, hydrophobic coat. Opposite to 'normal phase' HPLC, with RP-HPLC retention
time is longer for non-polar analytes and can be further increased by adding more water to
the mobile phase and decreased by adding more organic solute (e.g. methanol, acetoni-
trile). Analytes with a large number of non-polar C-H or C-C bonds have longer retention
times while those with polar
e
OH, -NH
2
or -COO
groups have short retention times.
Similarly, retention times decrease with double bond content (C-C single bond
e
C
double bond).
Table 13.3
presents relative retention times in RP-HPLC of various phospho-
lipids in the most commonly used mobile phases acetonitrile/water and propan-2-ol/
water.
>
C
ΒΌ
Fatty Acid Analysis
As discussed above, the various membrane lipid and phospholipid classes can be isolated,
primarily by TLC and RP-HPLC. Most membrane lipids are classified as 'complex lipids' (see
Chapter 4) that contain esterified fatty acids. Often the nature of these fatty acids constitutes
